Coccidioidomycosis is a systemic fungal infection common to southwestern United States. Most infections are self-limiting, but some patients develop disseminated coccidioidomycosis or persistent disease, either one of which can be life threatening. Our focus in the proposed research is the characterization of immunoreactive macromolecules of C. immitis. This includes antigens which stimulate T-cells as well as antibody response. The logic for this integrated approach is based on our current knowledge of the immunological response of the host to coccidioidal infections. Coccidioidomycosis is a disease in which T-cell mediated immunity has been shown to play a critical role in host defense, but little is known of the precise nature of T-cell reactive macromolecules. Early detection of primary coccidioidomycosis is indicated by a precipitin antibody response to C. immitis antigen. Once again, however, the precise nature of the serologically-reactive macromolecule(s) is unknown. To address these problems we have used the approach of identifying candidate molecules of C. immitis which stimulate T-cell or antibody response by a combination of biochemical and recombinant DNA techniques. We will initially identify C. immitis macromolecules which stimulate cell-mediated immunity by using antigen-specific murine T-cell lines. Candidate molecules are obtained by fractionation of complex mixtures previously demonstrated to be immunoreactive in cellular immunoassays. In a second approach, emphasized in the proposed research, CDNA expression libraries derived from MRNA of saprobic and parasitic phases of C. immitis and constructed in lambda ZAP II are screened with antibody raised against the above immunoreactive mixtures. Identification of CDNA clones which encode immunoreactive fusion proteins can ultimately lead to isolation of genes that can be introduced into appropriate expression vectors for in vitro or in vivo production of T-cell reactive proteins. Our research on serodiagnosis of early coccidioidal infection has focused on a 120-Kda glycoprotein that consists of a 3-O-methylated heteromannan. The latter, which binds specific anti-C. immitis patient IgM, is apparently unique to carbohydrate fractions of this fungus among the systemic fungal pathogens. Our specific aims in this part of the project are to isolate, purify, and characterize the immunoreactive oligosaccharide subfractions(s) of the extracellular, 120-Kda glycoprotein for use in serodiagnosis of primary coccidioidomycosis. Our overall goals are to characterize specific antigens of C. immitis which elicit humoral and cellular immune responses in the hopes of improving diagnostic reagents, and eventually developing a vaccine against coccidioidomycosis.